Premature birth is a major public health problem that causes 70-80% of neonatal morbidity and mortality. To address this problem our research focuses on understanding the hormonal control of labor and in particular how the steroid hormone progesterone acting through the nuclear progesterone receptors (nPRs) PR-A and PR-B, controls uterine contractility. Our central hypothesis is that in human pregnancy progesterone promotes myometrial quiescence via nPR-mediated genomic pathways and that parturition involves functional withdrawal of nPR-mediated progesterone actions, which allows the pro-contraction pathways to prevail leading to labor. Our studies show that human parturition involves a pre-partum- and labor-associated increase in the myometrial PR-A/PR-B protein ratio due to increased expression of PR-A. We also found that an increased level of PR-A relative to PR-B represses PR-B-mediated progesterone responsiveness in myometrial cells. Those observations have led to the PR-A/PR-B hypothesis, which posits that a) progesterone promotes myometrial relaxation during most of human pregnancy via PR-B, which modulates the expression of a specific cohort of relaxatory genes, and b) the relaxatory effects of PR-B are blocked at parturition by PR-A. PR-A repression of PR-B activity could occur at the promoters of endogenous progesterone-responsive genes, particularly those that inhibit contraction. Alternatively, PR-A could control the expression of a distinct cohort of genes that augment contractility and oppose the relaxatory effects of PR-B. To test this hypothesis we must identify the genes affected by PR-A and PR-B in myometrial cells. However, the PR-A and PR-B gene targets in the human pregnancy myometrium are not known. To address this major knowledge gap, the proposed studies will elucidate the genomic actions and interactions of PR-A and PR-B at endogenous progesterone responsive genes in myometrial cells. The genes and promoter elements affected by PR-A and PR-B will be determined using microarray-based genome-wide expression and DNA location analyses in a myometrial cell line and myometrial tissue specimens. To experimentally control the levels of PR-A and PR-B we will develop a stable genetically modified immortalized myometrial cell model, hTERT-HMA/B cells, containing independent inducible PR-A and PR-B transgenes. This is a novel and powerful approach to determine the specific genomic actions of progesterone via PR-A and PR-B, alone and in combination, in a human myometrial cell context. Two inter-related Specific Aims are proposed. SPECIFIC AIM 1: Identify the genes controlled by PR-A and PR-B in myometrial cells. The genes affected (up- or down-regulated) by PR-A and PR-B, alone and in combination, in hTERT-HMA/B cells will be identified using microarray-based genome-wide expression profiling. Genes of interest will be chosen based on the extent to which expression is altered by each nPR and whether their products could influence contractility based on known function. Confirmatory studies of selected genes will be performed in myometrium obtained from caesarean deliveries to determine whether expression levels correlate with the PR-A/PR-B protein ratio, stage of gestation and labor status. SPECIFIC AIM 2: Identify gene promoters that bind PR-A and PR-B in myometrial cells. The gene promoters to which PR- A and PR-B bind in hTERT-HMA/B cells will be identified by nPR-chromatin immunoprecipitation (ChIP) followed by microarray-based genome-wide DNA location analysis (ChIP-on-chip). Outcomes will be analyzed to identify PR-A and PR-B binding elements that lie within the promoters of PR-A and PR-B responsive genes identified in Specific Aim 1. The promoters of those genes will be the subjects of future mechanistic studies to determine how PR-A and PR-B interact to mediate genomic progesterone actions in the human pregnancy myometrium. Successful completion of the proposed studies will advance understanding of how progesterone regulates the engine for human birth, and contribute to the knowledge base needed to address the problem of preterm birth. PUBLIC HEALTH RELEVANCE: Preterm birth is a major socioeconomic problem that affects 10-15% of pregnancies and causes 70-80% of neonatal mortality and morbidity. Despite advances in neonatal care that have improved survival outcomes, the confounding problems due to preterm birth have a profound negative impact on a preterm infant's future health. Clearly, the final phase of fetal development is best achieved in the womb environment rather than in the neonatal intensive care unit. To this end, we must prevent and/or suppress preterm labor. However, current therapies to suppress preterm labor are generally ineffective. To resolve this problem we must fill the knowledge-gaps that limit our capacity to develop effective treatments for preterm labor. Therefore, our long- term goal is to determine the hormonal interactions that control uterine contractility. This proposal seeks to determine the mechanism by which the steroid hormone progesterone promotes uterine relaxation for most of pregnancy. A clear understanding of this process may reveal novel therapeutic targets for the suppression of preterm labor and the prevention of preterm birth.